Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection

viruses

Article Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection

Ting-Chun Hung 1,2, Alagie Jassey 3, Chien-Ju Lin 4, Ching-Hsuan Liu 5,6, Chun-Ching Lin 1,4, Ming-Hong Yen 1,4,* and Liang-Tzung Lin 5,7,*

1 Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 807 Kaohsiung, Taiwan; [email protected] (T.-C.H.); [email protected] (C.-C.L.) 2 Department of Clinical Pathology, Chi Mei Medical Center, Tainan 710, Taiwan 3 International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] 4 School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan; [email protected] 5 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] 6 Department of Microbiology & Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada 7 Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan * Correspondence: [email protected] (M.-H.Y.); [email protected] (L.-T.L.); Tel: +886-7-312-1101 ext. 2665 (M.-H.Y.); +886-2-2736-1661 ext. 3911 (L.-T.L.) Received: 18 September 2018; Accepted: 25 November 2018; Published: 27 November 2018

Abstract: Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any signiﬁcant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identiﬁed RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent.

Keywords: HCV; Rhizoma coptidis; herbal medicine; antiviral; entry inhibition

1. Introduction Hepatitis C virus (HCV) is an important liver pathogen belonging to the Flaviviridae family with an enveloped positive single-stranded RNA genome. HCV has seven genotypes (genotype 1~7) and a genome size of about 9.6 kb which encodes a polyprotein that is approximately 3000 amino acids long. The polyprotein upon translation is processed by viral and host proteases to yield 10 matured protein including structural proteins, Core, E1, E2, and p7 ion channel, as well as non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B [1]. HCV entry into the host hepatocytes is mediated by interaction

Viruses 2018, 10, 669; doi:10.3390/v10120669 www.mdpi.com/journal/viruses Viruses 2018, 10, 669 2 of 12 with several notable cell surface and tight junction receptors/co-receptors including heparin sulfate proteoglycans (HSPG), cluster of differentiation 81 (CD81), low density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1), and occludin (OCLN) [2,3]. Additional factors that can influence viral entry include apolipoprotein E (ApoE), which is incorporated on infectious HCV virions [4], and can function as an exchangeable apolipoprotein between secreted ApoE-associated lipoproteins and the HCV lipoviroparticle (LVP) to enhanced HCV infection [5]. There are over 170 million HCV carriers worldwide. HCV infection can lead to chronic hepatitis, cirrhosis, and liver cancer, and there is still no effective vaccine against the virus. While the previous standard of care consisting of PEGylated-interferon (IFN)-α in combination with ribavirin is associated with several important drawbacks including severe side effects and low efficacy against HCV genotype 1, the recent introduction of the direct-acting antivirals (DAAs) targeting the viral non-structural proteins has substantially improved the sustained virological response (SVR) in the most difficult to treat genotype 1 patients [6]. However, the DAAs also have challenges including potential toxicity, especially from drug-drug interactions (DDIs). For instance, HCV protease inhibitors are at high risk for DDIs as they are known substrates and inhibitors of cytochrome P450 (CYP) 3A4 system and can interfere with the metabolism of other drugs including immunosuppressants (e.g. cyclosporine and tacrolimus) when co-administered in liver transplant setting [7]. Other drug-drug interactions from HCV DAAs include those with acid-suppression therapies (e.g. famotidine and omeprazole) or the human immunodeficiency virus (HIV) antiretroviral agents (e.g. Rilpivirine and Efavirenz), which have been shown to decrease the effectiveness of the HCV NS5A inhibitor Ledipasvir [8] and produce adverse drug reactions with the protease inhibitor Paritaprevir [7], respectively. In addition, due to the great genetic variability of HCV, selection of resistant mutants is becoming a challenge as a greater number of people are being treated in real-world settings, which can potentially lead to DAA failures [6,9]. Therefore, continuous identification of novel candidate drugs particularly with a different mode of action to improve the current therapeutic strategies is highly envisaged. Rhizoma Coptidis (RC) is the dried rhizome typically obtained from Coptis chinensis Franch (‘Chinese goldthread’), which is a medicinal plant of the Ranunculaceae family [10]. RC is one of the most commonly used Chinese medicinal herbs (also known as ‘Huang Lian’) documented by the WHO [10] and is known to contain various bioactive alkaloids [11]. It is traditionally used for its “heat clearing” and “detoxification” effects such as treatment against arthritis, burns, eczema, microbial infections, and gastrointestinal diseases [10,12,13]. In addition, RC’s traditional usage against infections has been correlated through several recent studies that validated its antimicrobial functions, including against several bacteria and viruses [13]. Specifically, RC and its major constituents have been shown to exert inhibitory effects against herpesvirus, respiratory syncytial virus, and mouse hepatitis A virus infections [14–16]. These precedents suggest that RC may be a potent source for the discovery of novel antiviral treatments. Since the effect of RC on HCV infection remains largely unexplored, and in an attempt to identify novel anti-HCV agents, in this study we examined the impact of the methanolic extract of RC on HCV infection. Our results demonstrated that RC could robustly inhibit HCV infection by targeting the early steps in viral entry. Specifically, the targeted steps included viral attachment and entry/fusion into the host cells. In addition, the RC-mediated inhibition of HCV is not genotype-specific as the drug equally inhibits other HCV genotypes, thereby identifying RC as a potential pan-genotypic anti-HCV agent.

2. Materials and Methods

2.1. Cell Culture and Virus Production Culture of Huh-7.5 cells (human hepatoma, Huh-7 cell derivative) and production of cell-culture derived HCV particles (HCVcc) from the Gaussia luciferase reporter-tagged Jc1FLAG2(p7-nsGluc2A) construct (genotype 2a; kindly provided by Dr. Charles M. Rice) were carried out as previously described [17]. Virus concentration was expressed as multiplicity of infection (MOI) and the Viruses 2018, 10, 669 3 of 12 basal media for all viral infection analyses consisted of Dulbecco’s Modiﬁed Eagle’s Medium (GIBCO-Invitrogen, Carlsbad, CA, USA) containing 2% fetal bovine serum.

2.2. Plant Extract Preparation Rhizoma Coptidis roots from Coptis chinensis Franch (ID#kew-2736105 from The Plant List [18]) were obtained from local pharmacy store (Kaohsiung, Taiwan) and authenticated by Dr. Ming-Hong Yen using anatomical methods as well as by HPLC analysis through comparison to known molecular standards as previously described [10,19]. A voucher specimen was deposited at the Kaohsiung Medical University herbarium (CTM-RCC03). For methanol extraction [20], the roots were washed, dried, and homogenized before extraction with 100 % methanol, followed by concentration in vacuo. The methanolic RC stock was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) prior to use.

2.3. Cytotoxicity Assay and Antiviral Activity Analysis Huh-7.5 cells seeded at 1 × 104 cells/well in 96-well plates overnight were treated with increasing concentrations of RC for 5 days. The cells were then washed twice with phosphate buffered saline (PBS) before XTT cell viability analysis as previously described [21]. For examining antiviral activity, Huh-7.5 cells (1 × 104 cells/well in 96-well plates) were concurrently treated with the virus (MOI = 0.01) and the test drug at various concentrations before incubation at 37 ◦C for 3 days. The anti-HCV activity was determined by measuring the luciferase reporter signals using the BioLux™ Gaussia Luciferase Assay Kit (New England Biolabs; Pickering, ON, Canada) and a luminometer (Promega; Madison, WI, USA) as previously reported [22]. Data were expressed as percent (%) HCV infectivity from test treatments relative to medium control (virus only). IFN-α (Sigma) served as positive control.

2.4. Time-of-Drug-Addition Assay The time-of-drug-addition assay which provides information on the target of test agents in the viral life cycle consisted of pre-treatment, co-addition treatment, and post-infection treatment, and was performed as previously described [22]. For all analyses, Huh-7.5 cells were seeded in 96-well plates at a density of 1 × 104 cells/well overnight and infection with HCVcc was carried out at MOI = 0.01. Luciferase activity for all conditions was determined as described earlier following 72 h of incubation at 37 ◦C.

2.5. Synchronized Infection Analysis on Early Viral Entry The synchronized infection analysis to determine the effect of test agents on early viral entry was performed as previously described [22]. To examine viral inactivation, the test drug was incubated with the cell-free virus particles prior to diluting the virus-drug mixture to a subtherapeutic concentration and infecting the host cells (ﬁnal HCVcc MOI = 0.01). To investigate the inﬂuence on viral attachment, pre-chilled Huh-7.5 cells were challenged with the HCVcc (MOI = 0.01) in the presence/absence of the test agent at 4 ◦C, which allows binding of the viral particles to the host cells while precluding entry. To test for impact on viral entry/fusion, Huh-7.5 cells were pre-bound with HCVcc (MOI = 0.01) at 4 ◦C and then shifted to 37 ◦C incubation in the presence/absence of the test drug. For all the above analyses, Huh-7.5 cells were seeded in 96-well plates (1 × 104 cells/well) and luciferase activity for all conditions was determined as described earlier following 72 h of incubation at 37 ◦C.

2.6. Binding Assay The enzyme-linked immunosorbent assay (ELISA)-based binding assay was performed as previously described [23]. Brieﬂy, pre-chilled Huh-7.5 cells were challenged with HCVcc in the presence/absence of test drug at 4 ◦C for 3 h before washing with PBS and ﬁxing the cells with 4 % paraformaldehyde. Cell-bound virus was detected using primary anti-HCV E2 Viruses 2018, 10, 669 4 of 12 antibody (1:20000; AUSTRAL Biological, San Ramon, CA, USA) and secondary goat anti-mouse IgG conjugated with horseradish peroxidase antibody (1:36000, Invitrogen), followed by assessment with TMB (3,3’,5,5’-tetramethylbenzidine) Two-component Microwell Peroxidase Substrate Kit (KPL; Gaithersburg, MD, USA) and absorbance reading at 450 nm with a ELx800 microplate reader (Instrument, Inc.; Winooski, VT, USA).

2.7. Analysis Using HCV Pseudoparticles Retroviral pseudoparticles bearing HCV glycoproteins E1/E2 were produced following a previously described method [24] with some modifications. A pcDNA3.1 plasmid vector (Invitrogen) containing complete HCV core and E1/E2 glycoproteins of the HC-J6CH strain (genotype 2a; NC_009823) was co-transfected in conjunction with the pNL.Luc.Env−R+ construct (Env-defective retroviral backbone encoding firefly luciferase; kindly provided by Dr. Éric A. Cohen) into 293T cells using OMNIfect™ (transOMIC Technologies Inc.; Huntsville, AL, USA). Supernatant containing the viral pseudoparticles was harvested and filtered (0.45 µm) before being concentrated using 25% (v/v) polyethylene glycol and stored at −80 ◦C before use. For the infectivity experiment, the HCV pseudoparticles (HCVpp) were used to inoculate Huh-7.5 cell monolayers in 12-well plates in the presence or absence of the test agent for 2 h at 37 ◦C. The cells are then washed with PBS and further incubated in basal media for 72 h before being harvested and assessed for reporter luciferase activity using the Firefly Luciferase Assay kit (Promega) and a luminometer. Viral infectivity was calculated as percent (%) relative light units (RLU) compared to control (virus only).

2.8. Western Blot Cells were lysed with RIPA buffer supplemented with protease inhibitors (Roche Molecular Biochemicals; Indianapolis, IN, USA) and subjected to standard immunoblotting using anti-CD81 (1:1000; BD Biosciences, San Jose, CA, USA), anti-CLDN-1 (1:1000; Invitrogen), anti-SR-BI (1:1000; Abcam, Cambridge, UK), anti-OCLN (1:200; Cell Signaling Technology, Danvers, MA, USA), anti-Apolipoprotein B (ApoB, 1:5000; Abcam), anti-ApoE (1:5000; Calbiochem-Millipore, Billerica, MA, USA), and anti-β-actin (1:20000; Santa Cruz Biotechnology, Dallas, Texas, USA) primary antibodies, and anti-mouse (1:5000; Invitrogen) or anti-rabbit (1:2500; Sigma) secondary antibodies. Imaging was performed on an UVP chemiluminescence imaging system (UVP; Upland, CA, USA).

2.9. Inhibitory Effects Against Multiple Genotypes of HCV Antiviral activity against recombinant HCV carrying glycoproteins from genotype 2b (J8/JFH1), 3a (S52/JFH1), and 7a (QC69/JFH1) viruses (kindly provided by Dr. Jens Bukh) [25,26] was performed using a previously reported method [23] with some modiﬁcations. Huh-7.5 cells (1 × 104 cells/well in 96-well plates) were challenged with the viruses at MOI = 0.01 in the presence/absence of the test agent. After 3 days of further incubation, detection of viral infectivity was performed by immunoﬂuorescence staining of HCV foci using primary mouse anti-core clone B2 antibody (1:200; Anogen; Mississauga, ON, Canada), which cross-reacts different HCV core antigenic determinants, and a secondary Alexa Fluor 488 goat anti-mouse IgG (H+L) (1:400; Invitrogen) [27]. HCV-positive foci were quantitated and results were plotted against the DMSO control [28].

2.10. Statistical Analysis Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or unpaired t test. A p value of less than 0.05 (p < 0.05) was considered to be statistically signiﬁcant. All data are expressed as means ± standard error of the means (SEM) from three independent experiments. Viruses 2018, 10, 669 5 of 12

3. Results Viruses 2018, 10, x FOR PEER REVIEW 5 of 12 3.1. RC is Capable of Inhibiting HCV Infection Given the numerous numerous pharmacological properties of RC, we hypothesized that the medicinal medicinal herb herb could potentially possesspossess antiviral antiviral activities activities against against HCV. HCV. To To explore explore this this possibility, possibility, Huh-7.5 Huh cells‐7.5 werecells wereinfected infected with Gaussia with luciferaseGaussia luciferase reporter-tagged reporter HCVcc‐tagged in theHCVcc presence in the of differentpresence concentrations of different concentrationsof the methanolic of the extract methanolic of RC and extract the of luciferase RC and activitythe luciferase was subsequently activity was subsequently assessed to determine assessed toviral determine infectivity. viral A cytotoxicityinfectivity. A assay cytotoxicity was simultaneously assay was simultaneously performed on performed the cells with on the the same cells drugwith theconcentrations. same drug Ourconcentrations. results showed Our that results RC could showed inhibit that HCV RC infection could dose-dependentlyinhibit HCV infection and up dose to 50‐ dependently and up to 50 μg/mL without inducing significant cytotoxicity (Figure 1A). The 50 % µg/mL without inducing signiﬁcant cytotoxicity (Figure1A). The 50 % cytotoxic concentration (CC 50), cytotoxic concentration (CC50), the 50 % effective concentration (EC50), and the selective index (SI = the 50 % effective concentration (EC50), and the selective index (SI = CC50/EC50) were determined to CCbe 168.950/EC50±) were1.06 µ determinedg/mL, 20.07 to± be1.08 168.9µg/mL,  1.06 and μg/mL, 8.42, respectively20.07 ± 1.08 μ (Figureg/mL,1 B).and Since 8.42, the respectively antiviral (Figureefﬁcacy 1B). of RC Since appeared the antiviral to be comparableefficacy of RC between appeared 20 andto be 50 comparableµg/mL, a concentration between 20 and of 50 20 μµg/mL,g/mL awas concentration chosen for theof 20 remainder μg/mL was of our chosen experiments. for the remainder of our experiments.

Figure 1. Analysis of RC’s antiviral activity against HCV infection in Huh-7.5 hepatoma cells. Figure(A) Dose-response 1. Analysis analysis of RC’s of antiviral the cytotoxicity activity (relativeagainst toHCV mock infection control) in and Huh antiviral‐7.5 hepatoma efficacy (relative cells. (A to) Dose‐response analysis of the cytotoxicity (relative to mock control) and antiviral efficacy (relative to medium control) of RC against HCV infection; IFN-α (800 IU/mL) served as positive control. (B) CC50, medium control) of RC against HCV infection; IFN‐α (800 IU/mL) served as positive control. (B) CC50, EC50, and SI values determined from A. Data represent means ± SEM from 3 independent experiments. EC50, and SI values determined from A. Data represent means ± SEM from 3 independent 3.2. RCexperiments. Inhibits the Early Steps of HCV Entry In order to narrow down the window of antiviral activity from RC, we performed a 3.2. RC Inhibits the Early Steps of HCV Entry time-of-drug-addition assay wherein the drug was either added to cells 24 h before HCVcc infection (“pre-treatment”),In order to narrow concurrently down the added window at the of time antiviral of viral activity infection from (“co-addition”), RC, we performed or added a time after‐of‐ druginfection‐addition (“post-infection”) assay wherein and the incubated drug was for either 3 days added before to measuringcells 24 h before the luciferase HCVcc reporterinfection activity. (“pre‐ treatment”),Results indicated concurrently no significant added difference at the time in of HCV viral infectivity infection (“co in the‐addition”), presence or or absenceadded after of RC infection during (“postpretreatment‐infection”) (Figure and2 ),incubated suggesting for that 3 days the before drug does measuring not appear the luciferase to modulate reporter the host activity. cells Results before indicatedHCV infection. no significant In contrast, difference RC significantly in HCV inhibitedinfectivity HCV in the infectivity presence when or absence simultaneously of RC during added pretreatmentduring the viral (Figure challenge 2), suggesting as indicated that by the substantialdrug does not drop appear in the luciferaseto modulate signal the (Figure host cells2). Onlybefore a HCV infection. In contrast, RC significantly inhibited HCV infectivity when simultaneously added during the viral challenge as indicated by the substantial drop in the luciferase signal (Figure 2). Only a moderate decrease in viral infectivity was observed when the drug was added after the establishment of viral infection. In contrast, IFN‐α, which served as positive control, effectively inhibited the HCVcc infection in all 3 types of treatment. Thus, RC’s anti‐HCV activity appeared

Viruses 2018, 10, 669 6 of 12 moderate decrease in viral infectivity was observed when the drug was added after the establishment Virusesof viral 2018 infection., 10, x FOR PEER In contrast, REVIEW IFN-α, which served as positive control, effectively inhibited6 of the 12 HCVcc infection in all 3 types of treatment. Thus, RC’s anti-HCV activity appeared strongest when strongest when concurrently present on the host cell with the viral particles, suggesting that its concurrently present on the host cell with the viral particles, suggesting that its inhibitory effect mainly inhibitory effect mainly targeted the early phase of the HCV infection, including viral entry. targeted the early phase of the HCV infection, including viral entry.

Figure 2. Time-of-drug-addition analysis of RC’s antiviral effect. All data represent means ± SEM from Figure3 independent 2. Time‐ experiments.of‐drug‐addition RC =analysis 20 µg/mL; of RC’s DMSO antiviral = 0.5%; effect. IFN- Allα =data 800 represent IU/mL; * meansp < 0.05, ± SEM**p < from 0.01, 3ns: independent not significant. experiments. RC = 20 μg/mL; DMSO = 0.5%; IFN‐α = 800 IU/mL; *p < 0.05, **p < 0.01, ns: not significant. 3.3. RC Blocks HCV Viral Attachment and Entry/Fusion into the Host Cells 3.3. RCTo Blocks further HCV characterize Viral Attachment the mechanism(s) and Entry/Fusion underlying into the RC’s Host anti-HCV Cells effect, which was strongest whenTo RC further was characterize simultaneously the mechanism(s) present with underlying the virus on RC’s the anti host‐HCV cell effect, surface, which we was performed strongest a whensynchronized RC was infection simultaneously assay on earlypresent viral with entry. the To virus test whether on the RChost could cell inactivatesurface, we the performed cell-free viral a particles,synchronized the drug infection was pre-incubatedassay on early withviral the entry. HCVcc To test for whether 3 h before RC dilution could inactivate and infecting the Huh-7.5cell‐free viralcells. particles, The drug-virus the drug mixture was pre was‐incubated then diluted with 20Xthe HCVcc to yield for a non-effective 3 h before dilution concentration and infecting which Huh was‐ 7.5subsequently cells. The drug added‐virus to the mixture cells and was incubated then diluted for 20X 3 days. to yield Luciferase a non‐effective readings concentration following 3 days which of wasincubation subsequently showed added no significant to the cells difference and incubated between for samples 3 days. treated Luciferase with readings or without following RC, suggesting 3 days ofthat incubation the drug does showed not impact no significant the free viral difference particles between (Figure3 A).samples To determine treated the with effect or of without RC on viral RC, ◦ suggestingattachment, that we specificallythe drug does added not RCimpact during the HCV free viral cell binding particles at (Figure 4 C, which 3A). allowsTo determine for virus the binding effect ofbut RC precludes on viral viralattachment, internalization, we specifically and then added tested RC the during reporter HCV readout cell binding at the endat 4 of°C, the which incubation. allows forResults virus demonstrated binding but precludes a significant viral decrease internalization, in the luciferase and then reporter tested the activity, reporter which readout indicated at the thatend ofRC the interfered incubation. with Results the attachment demonstrated of the virus a significant to the host decrease cells (Figure in the3A). luciferase To ascertain reporter whether activity, RC whichinfluenced indicated the post-attachment that RC interfered viral with entry/fusion the attachment step, Huh-7.5 of the virus cells to were the pre-boundhost cells (Figure with HCV 3A). at To 4 ◦ ◦ ascertainC before whether shifting the RC temperature influenced the to 37postC‐attachment in the presence viral of entry/fusion RC. Similar tostep, the Huh effect‐7.5 observed cells were on viralpre‐ boundattachment, with HCV our data at 4 revealed°C before a shifting significant the temperature decrease in HCV to 37 infectivity°C in the presence at the end of RC. of the Similar incubation, to the effectsuggesting observed that on RC viral equally attachment, blocked HCVour data entry/fusion revealed a (Figure significant3A). decrease in HCV infectivity at the end of the incubation, suggesting that RC equally blocked HCV entry/fusion (Figure 3A).

Viruses 2018, 10, 669 7 of 12 Viruses 2018, 10, x FOR PEER REVIEW 7 of 12

Figure 3. Investigation of RC’s antiviral effect on HCV entry. (A) Synchronized infection analysis ofFigure RC treatment 3. Investigation on HCV of early RC’s viralantiviral entry. effect (B) on Validation HCV entry. of RC’s (A) Synchronized inhibitory activity infection against analysis HCV of attachmentRC treatment using on ELISA-based HCV early virus viral binding entry. assay.(B) Validation (C) Impact of of RC’s RC treatment inhibitory on activity HCVpp against infection HCV of Huh-7.5attachment cells. using (D) Western ELISA‐based blot analysis virus binding of RC treatmentassay. (C) effectImpact on of HCV RC treatment host cell entryon HCVpp factors. infection Data representof Huh‐7.5 means cells.± (DSEM) Western from 3 blot independent analysis of experiments. RC treatment RC effect = 20 µong/mL HCV unless host cell otherwise entry factors. indicated; Data DMSOrepresent = 0.5 means %; *p <± 0.05,SEM **fromp < 0.01,3 independent ***p < 0.001. experiments. For Western RC blot = 20 analysis, μg/mL representative unless otherwise blots indicated; from 3 independentDMSO = 0.5 experiments %; *p < 0.05, are**p shown.< 0.01, ***β-actinp < 0.001. served For asWestern loading blot control. analysis, representative blots from 3 independent experiments are shown. β‐actin served as loading control.

Viruses 2018, 10, 669 8 of 12

3.4. Confirmation of RC’s Antagonism Against HCV Cell Attachment by ELISA-Based Virus Binding Assay To directly validate our finding that RC inhibits HCV attachment to the host cells, we employed an ELISA-based binding assay. Huh-7.5 cells were inoculated with HCVcc in the presence of RC at 4 ◦C for 3 h, after which the cells were washed, fixed, and subjected to colorimetric analysis by detecting cell surface-bound HCV particles using anti-HCV E2 antibody. As depicted in Figure3B, RC treatment dose-dependently decreased viral attachment as demonstrated by the sharp decline in the absorbance signal, which is in agreement with the above results.

3.5. RC Robustly Inhibits Infection by Pseudoparticles Bearing HCV Glycoproteins HCV entry steps are mediated by viral glycoproteins [2]. Based on the inhibitory effects observed from RC against HCV entry steps, and to validate its antiviral potency against HCV entry, we further examined the impact of RC treatment on infection by retroviral pseudoparticles bearing HCV glycoproteins E1/E2 (HCVpp). Speciﬁcally, luciferase-tagged HCVpp were used to infect Huh-7.5 cells in the presence or absence of RC, before further incubating the cells and assessing luciferase reporter activity as a readout of viral infectivity. As demonstrated in Figure3C, RC robustly suppressed HCVpp infection of the Huh-7.5 hepatoma cells compared to the DMSO control. This result therefore conﬁrms and validates the antiviral capacity of RC against the viral entry stage of HCV.

3.6. RC Does Not Modulate Host Cell Entry Factors to HCV Infection HCV infection of hepatocytes is a well-orchestrated process involving the engagement of several host factors including CD81, SR-BI, CLDN-1, and OCLN. In addition, ApoE has also been reported to play a role in mediating HCV entry [5]. Given that RC treatment blocked HCV infection mainly by targeting viral entry, we next asked whether the drug could modulate the expression of key host cells entry factors. To this end, Huh-7.5 cells were pre-treated with RC for 24 h before harvesting the cell lysates for Western blot analysis. As depicted in Figure3D, pre-treatment of Huh-7.5 cells with RC did not alter the expression of CD81, SR-BI, CLDN-1, and OCLN. Similarly, the expression of ApoE was not affected by RC treatment (Figure3D). ApoB, which participates in HCV virion release was included for comparison and was neither affected by the drug treatment. These results therefore suggested that RC did not inhibit HCV infection via inﬂuencing the host cells and is consistent with our data from the pre-treatment analysis (Figure2).

3.7. RC Inhibits Multiple HCV Genotypes Since RC appeared to primarily target HCV entry, we finally sought to examine whether the medicinal herb also possesses a pan-genotypic activity against HCV. For this purpose, Huh-7.5 cells were seeded and infected with recombinant HCVcc expressing glycoproteins from genotypes 2b (J8/JFH1), 3a (S52/JFH1), and 7a (QC69/JFH1) in the presence of the RC before further incubation and analysis of HCV infectivity by immunofluorescence staining. Results showed a significant decrease in viral infection across all the tested HCV genotypes when RC was present, suggesting that it may possess a pan-genotypic activity against HCV (Figure4). VirusesViruses 20182018,, 1010,, 669x FOR PEER REVIEW 9 of 12

Figure 4. Inhibitory effect of RC treatment on multiple HCV genotypes. All data represent means ± SEMFigure from 4. Inhibitory 3 independent effect experiments. of RC treatment RC = on 20 multipleµg/mL; DMSOHCV genotypes. = 0.5 %; *** Allp < data 0.001, represent unpaired meanst test. ± SEM from 3 independent experiments. RC = 20 μg/mL; DMSO = 0.5 %; ***p < 0.001, unpaired t test. 4. Discussion and Conclusions 4. DiscussionContinuous and identification Conclusion of novel antivirals with various modes of action is important given that developmentContinuous identification of drug resistance of novel is antivirals commonplace with especiallyvarious modes with of viruses action that is important exhibit genetic given variability,that development including of HCV. drug In resistance this study, is we commonplace demonstrated especially for the first with time viruses that the that methanolic exhibit genetic extract ofvariability, RC robustly including inhibited HCV. HCV In infection. this study, Specifically, we demonstrated RC mainly for targetedthe first thetime HCV that early the methanolic viral entry stepsextract such of RC as robustly attachment inhibited and entry/fusion HCV infection. to theSpecifically, host cells. RC Interestingly, mainly targeted RC alsothe HCV inhibited early HCV viral infectionentry steps of such several as otherattachment genotypes. and entry/fusion Our results therefore to the host identified cells. Interestingly, RC as a promising RC also antagonist inhibited HCV with pan-genotypicinfection of several function other against genotypes. HCV entry,Our results which therefore could be usefulidentified for developing RC as a promising HCV prophylaxis. antagonist with Currentlypan‐genotypic no approved function therapeutic against HCV treatment entry, exists which for could targeting be HCVuseful entry, for anddeveloping patients HCV with chronicprophylaxis. hepatitis C are at risk for end-stage liver diseases such as cirrhosis and liver cancer, which necessitateCurrently liver no transplantation. approved therapeutic Importantly, treatment donor exists livers for inadvertently targeting HCV become entry, re-infected and patients almost with immediatelychronic hepatitis after C transplantation are at risk for end in hepatitis‐stage liver C patientsdiseases [ 29such]. Given as cirrhosis that the and DAAs liver incancer, current which use cannotnecessitate prevent liver liver transplantation. graft re-infection Importantly, and have donor the propensity livers inadvertently to select for become drug-resistant re‐infected mutants, almost combiningimmediately entry after inhibitors transplantation with the in DAAshepatitis would C patients be expected [29]. Given to broaden that the the DAAs treatment in current strategies use againstcannot prevent hepatitis liver C especially graft re‐infection in the liver and transplanthave the propensity setting. Interestingly, to select for drug previous‐resistant studies mutants, have demonstratedcombining entry that inhibitors combining with DAAs the DAAs and virus would entry be inhibitorsexpected to can broaden produce the a treatment synergistic strategies effect to improveagainst hepatitis drug efficacy C especially [30]. Our in the discovery liver transplant that RC can setting. antagonize Interestingly, HCV entry previous makes studies it an idealhave candidatedemonstrated for usethat in combining liver transplant DAAs scenarios and virus and entry to testinhibitors in combination can produce with a thesynergistic DAAs for effect better to therapeuticimprove drug efficacy. efficacy [30]. Our discovery that RC can antagonize HCV entry makes it an ideal candidateNatural for resourcesuse in liver such transplant as plant scenarios materials and serveto test as in excellentcombination starting with the point DAAs for for antiviral better discoverytherapeutic [31 efficacy.]. RC’s anti-HCV bioactivity identifies the medicinal herb as an important antiviral sourceNatural for the resources treatment such of as hepatitis plant materials C. Various serve medicinalas excellent plant starting extracts point for have antiviral been showndiscovery to possess[31]. RC’s anti-HCV anti‐HCV properties bioactivity and identifies thereafter the served medicinal as source herb as for an further important identifying antiviral small source molecule for the inhibitors.treatment of Examples hepatitis of C. those Various that specificallymedicinal plant target extracts HCV entry have include been shown silibinin to and possess silymarin anti‐HCV from Silybumproperties marianum and thereafter[32,33 ],served gallic as acid source found for infurtherLimonium identifying sinense small[22], molecule loliolide inhibitors. and the butenolide Examples (4R,6S)-2-dihydromenisdaurilideof those that specifically target HCV derived entry from includePhyllanthus silibinin urinaria and silymarin[28,34], from ladanein Silybum isolated marianum from Marrubium[32,33], gallic peregrinum acid foundL. (Lamiaceae) in Limonium [35], andsinenseBupleurum [22], kaoiloliolideand its and terpenoid the butenolide saikosaponin (4R,6S) b2 [23‐2].‐ Whiledihydromenisdaurilide the molecular constituents derived from in RC Phyllanthus contributing urinaria to its [28,34], anti-HCV ladanein activity isolated remain from to be Marrubium identified, theperegrinum alkaloids L. (Lamiaceae) are potential [35], candidate and Bupleurum bioactives kaoi which and its are terpenoid known to saikosaponin be the major b2 components [23]. While the of RCmolecular and are constituents typically present in RC in contributing the alcoholic to extracts its anti of‐HCV the herb activity [11]. remain Examples to includebe identified, berberine, the coptisine,alkaloids are palmatine, potential epiberberine, candidate bioactives columbamine, which are jatrorrhizine, known to be and the groenlandicine, major components among of RC other and majorare typically compounds present [11 in]. the Whether alcoholic a single extracts compound of the herb or [11]. a combination Examples include of them berberine, contributes coptisine, to RC’s antiviralpalmatine, actions epiberberine, against HCV columbamine, remains to be jatrorrhizine, explored. Further and in-depthgroenlandicine, analysis wouldamong be other required major to fullycompounds elucidate [11]. the Whether bioactive a ingredientssingle compound responsible or a combination for RC’s anti-HCV of them effect. contributes to RC’s antiviral actionsOur against results HCV indicated remains that RCto be specifically explored. blocked Further HCV in‐depth attachment analysis and would entry/fusion be required into theto fully host cellselucidate without the bioactive significantly ingredients influencing responsible the cell-free for RC’s viral anti particles‐HCV effect. or modulating key host cell entry factorsOur to results HCV. The indicated ability that of RC RC to specifically inhibit HCVpp blocked infection HCV suggestsattachment that and its mechanisticentry/fusion target(s)into the host cells without significantly influencing the cell‐free viral particles or modulating key host cell

Viruses 2018, 10, 669 10 of 12 may involve the HCV glycoproteins-mediated interactions with the host cell. A potential mechanism is the transient interaction(s) with the HCV glycoproteins that are inadequate to inactivate free viral particles but sufﬁciently effective to hinder contact with cell surface receptors/co-receptors. Additional mechanisms of action could also include concentration-dependent reversible conformational alterations in receptors/co-receptors or viral particles’ structure to block viral entry. We also noted a moderate effect from RC in the post-infection stage (Figure2), albeit less pronounced compared to its impact in inhibiting HCV entry steps. This effect was not entirely due to inhibition of entry in subsequent de novo infection cycles, since treatment of subgenomic HCV replicon cells with RC also showed a moderate decrease in viral RNA in a preliminary experiment (Figure S1). It is possible that RC may possess multiple mechanisms against HCV infection, which would necessitate further studies for clariﬁcation. Nonetheless, our results clearly demonstrated that RC most potently inhibited HCV entry. Therefore, we suggest that RC could be further explored for prophylactic/therapeutic management of hepatitis C.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/10/12/669/ s1, Figure S1: Effect of RC treatment on HCV subgenomic replicon cells. Author Contributions: Conceived and designed the experiments: T.-C.H., L.-T.L. Performed the experiments: T.-C.H., A.J., C.-J.L., C.-H.L. Supervised all research: M.-H.Y., L.-T.L. Analyzed the data: T.-C.H., A.J., C.-J.L., C.-H.L., C.-C.L., M.-H.Y., L.-T.L. Wrote and edited the paper: T.-C.H., A.J., C.-H.L., M.-H.Y., L.-T.L. All authors contributed to reagents/materials/technical support to this study. Funding: This study was supported in part by funding from the Ministry of Science and Technology of Taiwan (MOST104-2320-B-037-011 to C.-C.L. and MOST107-2320-B-038-034-MY3 to L.-T.L.). Acknowledgments: The authors would like to thank Charles M. Rice, Jens Bukh, and Éric A. Cohen for reagents, Christopher D. Richardson for experimental support for the antiviral assay against the various HCV genotypes, and Shun-Pang Chang and Chueh-Yao Chung for technical assistance. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

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